1. Field of the Invention
The present invention relates generally to methods and compositions for production of amino acids by bacteria of the genus Corynebacterium or Brevibacterium using sucrose as a carbon source. More particularly, the invention relates to the production of L-lysine from Corynebacterium glutamicum using sucrose as a carbon source.
2. Related Art
Many commercial fermentations of Corynebacterium and Brevibacterium use glucose as a carbon source. Consequently, many bacterial production cultures have been designed to optimize rates of production and/or yields using glucose as a carbon source. Production of the commercially important amino acid L-lysine was been a particular target of optimization.
Because of cost and other possible considerations, use of alternative, non-glucose carbon sources may be preferred in some parts of the world. One possible non-glucose carbon source is sucrose. Sucrose may be obtained, for example, from sugar cane or sugar beet. Unfortunately, because microorganisms often transport and utilize sucrose differently than glucose, production of a desired amino acid or fine chemical product from many microorganisms using sucrose as a carbon source can suffer from reduced efficiency. This may particularly be the case where the microorganisms using sucrose as a carbon source have been designed for optimal growth on glucose. That is the case for Corynebacterium glutamicum, one of the microorganisms most commonly used for the manufacture of amino acids such L-lysine by fermentation.
A metabolic pathway for utilization of sucrose in C. glutamicum was suggested by Wittmann, et al., “Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source,” Appl. & Enviro. Microbiol. 70(12): 7277-7287 (2004). Wittmann, et al., hypothesized that Corynebacterium glutamicum has a sucrose uptake mechanism that occurs by a phosphotransferase system (PTS), in which sucrose is phosphorylated at the glucose ring and subsequently hydrolyzed in the cell to glucose-6-phosphate and fructose. The PTS in C. glutamicum is a general carbohydrate transfer system that utilizes of a combination of two commonly shared cytoplasmic proteins designated enzyme I and Hpr, encoded by the ptsI and ptsH genes respectively, that interact with a discrete set of membrane bound EII proteins complexes designated fructose-pts, sucrose-pts, and glucose-pts encoded by the ptsF, ptsS, and ptsG genes respectively, that preferentially transport fructose, sucrose and glucose, respectively (Tanaka et al, Microbiology (2008) 154, 264-274). There are also two pts genes designated HCg12933 and NCg12934 that encode proteins with unknown specificities (id.).
Wittmann, et al., also suggested that following the hydrolysis, the resulting fructose is excreted from the cell, then reimported through the fructose-PTS uptake system and the mannose PTS uptake system (the latter now believed to be the same as the glucose-PTS). The existence of multiple uptake systems for sucrose (sucrose-PTS, fructose-PTS, and glucose-PTS) and therefore multiple entry points for carbon into the cell has been hypothesized as a possible reason for unfavorable performance on sucrose.
During lysine production on glucose, about 65% of the carbon goes down the pentose phosphate pathway (PPP) for the production of NADPH, which is used in lysine synthesis. During lysine synthesis on sucrose, however, it is believed that a much lower percentage of carbon goes down the PPP, because slightly less than half of the total carbon enters glycolysis as fructose-1,6-phosphate.
As illustrated in FIG. 1, when a wild type C. glutamicum is grown on sucrose, it is believed that about 90% of the fructose that would enter the C. glutamicum cell enters through the fructose-PTS as fructose-1-phosphate. The fructose-1-phosphate is phosphorylated to make fructose-1,6-phosphate. It is believed that fructose-1,6-phosphate does not go through the PPP pathway, in large part because 6-phosphofructokinase is largely an irreversible enzyme and there is very little fructose-1,6-biphosphatase activity in C. glutamicum grown on sucrose. Fructose-1,6-diphosphate will therefore preferentially enter glycolysis and the TCA cycle, which does not provide reducing power for commercial level lysine synthesis.
About 10% of fructose entering C. glutamicum is believed to be taken up by the glucose-PTS system as fructose-6-phosphate. Fructose-6-phosphate may contribute to the amount of carbon directed to PPP flux by action of glucose-6-phosphate isomerase operating in the gluconeogenetic direction to produce glucose-6-phosphate from fructose-6-phosphate. One proposed metabolic pathway for sucrose utilization in Corynebacterium including routes through glycolysis and the PPP shunt is shown in FIG. 1.
Increased lysine production from Corynebacterium on sucrose has been evaluated. One method that might be used to increase production was set forth by Georgi, et al., “Lysine and Glutamate Production by Corynebacterium glutamicum on Glucose, Fructose, and Sucrose: Roles of Malic Enzyme and Fructose-1,6-bisphosphatase,” Metabolic Eng. 7:291-301 (2005).
The Georgi, et al., strategy purportedly involves use of a constitutive promoter to overexpress the fructose-1,6-bisphosphatase gene fbp. This strategy may be unfavorable, however, because it may result in the creation of strains of Corynebacterium that, while optimized for growth on sucrose, have characteristics that could lead to suboptimal growth on glucose. This may potentially reduce the flexibility of the economically viable uses of the strains, which may not perform in a way that allows use of either sucrose or glucose as a carbon source, depending on what is economically advantageous as conditions change.
Another purported strategy for increasing lysine production from Corynebacterium on sucrose is reported in WO2005/059139A2, as well as Becker, et al., “Amplified Expression of Fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose of phosphates pathway and of lysine production on different carbon sources,” Appl. Envir. Microbiol. 71: 8587-8596 (2005). In this strategy, fructose-1,6-bisphosphatase is overexpressed. This purportedly allows fructose-1,6-P to return to the PPP, eventually increasing the amount of NADPH. The basic strategy of Georgi, et al. and Becker, et al. is illustrated in FIG. 5.
A further proposal for possibly increasing lysine production from Corynebacterium using sucrose as a carbon source was reported in Moon, et al., “Analyses of enzyme II gene mutants for sugar transport and heterologous expression of fructokinase gene in Corynebacterium glutamicum ATCC 13032” FEMS Microbiol. Lett. 244: 259-266 (2005). Moon demonstrated that expression of a Clostridium acetoybutylicum fructose kinase gene in C. glutamicum reduced the fructose exported into the media from the transformed strain during growth on sucrose which was otherwise exported during log phase growth of the parent strain lacking the fructokinse activity. Moon, et al. also demonstrated that a ptsF mutant strain lacking fructose-pts activity but expressing the fructokinse enzyme was able to grow to a higher optical density and utilize exported fructose better than the mutant strain lacking the fructokinase activity. It was hypothesized that expression of fructokinase in C. glutamicum would allow the conversion of fructose- to fructose 6-P which would then proceed to the PPP shunt possibly increasing lysine production, instead of being exported from the cell and then re-imported via the PTS system predominantly as fructose 1-phosphate. The scheme hypothesized by Moon, et al. is illustrated in FIG. 6.
The kinetics of lysine production with fructose and sucrose as carbon sources is reported in Kiefer, et al., “Influence of glucose, fructose and sucrose as carbon sources on kinetics and stoichiometry of lysine production by Corynebacterium glutamicum,” J. Indus. Microbiol. & Biotech. 28: 338-343 (2002); and Kiefer, et al., “Comparative Metabolic Flux Analysis of Lysine-Producing Corynebacterium glutamicum Cultured on Glucose or Fructose,” Appl. & Envir. Microbiol. 70(1): 229-239 (2004). The inclusion of the E. coli xylose isomerase gene in an altered Corynebacterium cell is reported in Kawaguchi, et al., “Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum,” Appl. & Envir. Microbiol. 72(5): 3418-3428 (2006)
It would be desirable to create a strain of microorganism that is optimized for growth on sucrose but that retains characteristics favorable for growth on glucose. Such a strain could result in fermentative production of amino acids from sucrose being more economically favorable when sucrose is available; when sucrose is unavailable or is not as plentiful or inexpensive as glucose, such a strain could efficiently be used for fermentative production of amino acids from glucose. It is an object of the invention to create such a strain. It is also an object of the invention to produce lysine using strains produced by embodiments of the invention. Preferably, the amount and/or rate of lysine production will be greater in bacteria produced in embodiments of the invention. Of course, the invention as defined by the claims shall not be limited by its ability to satisfy one or more of the objects of the invention.